KR20000016389A - Exendin analogues, processes for their preparation and medicaments containing them - Google Patents

Abstract

PURPOSE: An exendin derivative, a manufacturing method thereof, and a medicament containing the derivative are provided to be used for treating diabetes mellitus. CONSTITUTION: The invention concerns novel exendin analogues which can be used in the treatment of diabetes mellitus. The invention also concerns processes for preparing these substances and medicaments containing them. The exendin analogues are derived from SEQ ID NO:1 (I) or SEQ ID NO:2 (II).

Description

Exendin analogs, preparation methods thereof, and pharmaceuticals containing the same

The present invention relates to novel exendin analogs that can be used in the treatment of diabetes mellitus, methods for their preparation, and medicaments containing the same.

Background of the Invention

After a functional link between the small intestine and the exocrine pancreas was demonstrated in the sixties, it became possible to measure insulin accurately in plasma. Insulin responses after oral glucose administration are much more potent than intravenous glucose administration, even if blood levels of the same glucose are reached. This "incretin effect" is explained by the functional combination of the entero-insular axis. It is released from the small intestine after meals, circulated in the plasma with marked levels of increase, and enterohormones that amplify glucose-induced insulin release contribute to this effect. In addition to the typical incretin hormone gastric inhibitory polypeptide I (GIP), glucagon-like peptide 1 (GLP-1) is currently attracting attention. In a relatively short time, GLP-1 became the most physiologically incredible hormone candidate for the potential alternative to the treatment of type II diabetes. The present invention describes new materials that mimic the effects of naturally occurring GLP-1 molecules. This new material is characterized by increased stability while maintaining efficiency.

Anti-diabetic pathogenic effect

Infusion and subcutaneous injection of GLP-1 cause a significant increase in insulin secretion and inhibition of glucagon release in patients with type II diabetes (Gutniak, M. (1992); Kreymann, B. (1987); Nathan, DM (1992) Nauck, MA (1993 a & b)). Both are related to treatment and to blood sugar that lowers the effects of GLP-1: insulin promotes glucose uptake by its target tissues and inhibits your life. GLP-1 analogs also increase glucose uptake in the periphery. Inhibition of glucagon secretion is considered an indirect GLP-1 effect because glucagon producing A cells do not express the GLP-1 receptor (Komatsu, R. (1989)). In contrast, increased insulin and somatostatin release show decisive factors. Both hormones are known as inhibitors of glucagon release.

Two molecular mechanisms certainly contribute to GLP-1 induced insulin increase in type II diabetes. In addition to directly amplifying glucose induced insulin release, GLP-1 sensitizes a subpopulation of B cells for main stimulation "glucose" and also possibly further stimulation (Fehmann, HC (1991)), resulting in more overall B Allow cells to secrete insulin. This priming effect is the most reasonable explanation for the fact that GLP-1 leads to prolonged release of insulin despite its relatively short plasma half-life.

This effect depends on increased glucose levels (> 108 mg / dl) (Goke, R. (1993a)). This is fundamentally different from sulfonylureas, which affect insulin secretion independently of GLP-1 and glucose blood systems. If the glucose level decreases below 108 mg / dl, insulin secretion stops despite intravenous infusion of GLP-1. Thus hypoglycemia is rarely expected when GLP-1 is used therapeutically. In fact, this is also not described in existing clinical studies. However, the pharmacodynamic characteristics of GLP-1 are problematic. The duration of action is limited due to its very short half-life.

The synthesis of stable and potent GLP-1 peptide analogs from a therapeutic point of view is preferred in any case. Currently peptide analogues have been synthesized based on the molecular exendin from which lizards were originally isolated from venom to develop improved therapeutics that are stable against degradation with increasing duration of therapeutic action in diabetes. This peptide has the same pharmacological effect as GLP-1, but surprisingly has a fairly long half-life.

The new peptide sequences described as subjects of the present invention have effects on insulin synthesis and insulin release and insulin effects, in particular on the uptake of glucose and fasting of the stomach into target tissues, muscle and adipose tissue.

Subject of the invention

The present invention is based on the sequence of peptides, exendin-3 and exendin-4 isolated from secretions of either Heloderma horridum or Heloderma suspectum (Eng. J.et al. (1990, 1992)). The effect of the two peptides on the amino acid sequence and the pancreas has already been published by several authors. (Eng. J. et al. (1990); Raufman, JP (1992); Goke, R. (1993b); Thorens, B (1993). Subject of the invention is a new nodular exendin fragment containing an amino acid sequence of exendin-3- (1-30) or exendin-4- (1-30), wherein the C-terminus of the sequence is three amino acids , Preferably at most one amino acid, and the N-terminus may be shortened to two or less, preferably at most one amino acid. Surprisingly, these exendin fragments are biologically activated even if the amino acid sequence is shortened. Shortened amino acid sequences are much more economical to produce than relatively long sequences. Thus, peptide fragments having the following sequence are particularly preferred; In particular peptide fragments based on exendin-3- (1-30) (SEQ ID NO: 1):

SEQ ID NO: 1 based on exendin-3

SEQ ID NO: 2 based on exendin-4

Here, the amino acids at positions 1, 2, 28, 29 or 30 may be part of a sequence which depends on the desired chain length. Peptides are numbered from the N-terminus to the C-terminus. X ₁ represents a proteogenic or nonproteinaceous amino acid except glycine.

Exendin and exendin analogs having chain lengths of 1-27 are preferred, especially analogues of 1-30 chain length.

The carboxyl group COR ₃ of the amino acid at the C-terminus may exist in free form (R ₃ = OH) or in the form of physiologically acceptable alkali or alkaline earth metals such as sodium, potassium or calcium salts. Carboxyl groups can also be esterified with primary, secondary or tertiary alcohols such as methanol, branched or unbranched C ₁ -C ₆ -alkyl alcohols, in particular ethyl alcohol or tert-butanol. However, the carboxyl groups can also be amidated with primary or secondary amines such as ammonia, branched or unbranched C ₁ -C ₆ alkylamines or C ₁ -C ₆ di-alkylamines, in particular methylamine or dimethylamine.

The amino group at amino acid NR ₁ R ₂ at the N-terminus may exist in free form (R ₁ , R ₂ = H) or in the form of a physiologically acceptable salt, such as chloride or acetate. The amino groups can also be acetylated with acids to be R ₁ = H and R ₂ = acetyl, trifluoroacetyl, adamantyl, or protected forms with common amino protecting groups of peptide chemistry such as Fmoc, Z, Boc, Alloc Present or N-alkylated (R ₁ and / or R ₂ = C ₁ -C ₆ alkyl or C ₂ -C ₈ alkenyl or C ₇ -C ₉ aralkyl).

By alkyl residue is meant straight, branched or any cyclic alkyl moiety, preferably methyl, ethyl, isopropyl and cyclohexyl.

All exendin fragments may exist as fully or partially protected derivatives.

A further subject of the invention is an exendin fragment having the above-described properties and a sequence length in which 1 to 11 alterations listed in (a) to (p) below are carried out. Exendin fragments are exendin fragments having up to 9, particularly preferably up to 5 alterations listed in (a) to (p) below.

(a) the α-amino acid at position 1 is D-His, Ala, D-Ala, Gly, Lys or D-Lys, of which Ala, Gly or Lys is particularly preferred; or

(b) α-amino acid at position 2 is Ser, D-Ser, Thr, D-Thr, Gly, Ala, D-Ala, Ile, D-Ile, Val, D-Val, Leu or D-Leu, preferably Preferably Ser, Thr, Gly, Ala, Ile, Val, or Leu; or

(c) the α-amanoic acid at position 3 is Glu, D-Glu, Asp, D-Asp, Ala or D-Ala, preferably Glu, Asp or Ala; or

(d) the amino acid at position 4 is Ala, D-Ala or β-Ala, preferably Ala; or

(e) the α-amino acid at position 5 is Ser, Tyr or Ala; or

(f) the α-amino acid at position 6 is Ala, Ile, Val, Leu, Cha or Tyr, preferably Ala, Ile, Val, Leu, or Tyr; or

(g) the α-amino acid at position 7 is Ala, D-Ala, Tyr, D-Tyr, Ser, D-Ser or D-Thr, preferably Ala, Tyr or Ser; or

(h) the α-amino acid at position 8 is Ala, Tyr, or Thr; or

(i) the α-amino acid at position 9 is Ala, D-Ala, Glu, D-Glu or D-Asp, preferably Ala or Glu; or

(j) amino acids at positions 10,11,12,15,16,17,18,19,20,21,24,28,29 are independently mutually proteinaceous or nonproteinaceous D- or L-amino acids, preferably Preferably a proteinaceous L-amino acid; or

(k) the α-amino acid at position 13 is a neutral L-amino acid, preferably a neutral proteinaceous L-amino acid; or

(l) α-amino acids at position 14 are neutral L- or D-amino acids (except L-leucine) for stabilization, preferably Nle, D-Nle, Ala, D-Ala, Ile, D-Ile, Val Or D-Val, with Ile, Val or Ala especially preferred; or

(m) α-amino acid at position 22 is D-Phe, Tyr, D-Tyr, Leu, D-Leu, Val, D-Val, L-Cha, D-Cha, β-1-Nal, β-2 -Nal or β-1-D-Nal, of which Tyr, Leu or Val are preferred; or

(n) the α-amino acid at position 23 is Leu, D-Leu, D-Ile, Val, D-Val, L-Cha, D-Cha, Tyr, D-Tyr, D-Tyr, Phe or D-Phe Of which Leu, Val, Tyr or Phe are preferred; or

(o) the α-amino acid at position 25, 26 or 27 is a neutral L- or D-amino acid, preferably a neutral, proteinaceous L-amino acid; or

(p) The α-amino acid at position 30 is particularly preferably proteinaceous or nonproteinaceous D- or L-amino acid (except glycine), preferably Arg, D-Arg, Tyr or D-Tyr, Arg or Tyr .

Among the new exendin fragments, in addition to having the above mentioned properties and arrangement lengths, amino acid leucine at position 10 and / or amino acid valine at position 19, amino acid isoleucine or alanine at position 14 instead of methionine, arginine at position 30 It is particularly preferred to have This modification of the exendin fragment is particularly preferred in addition to the above-mentioned particularly preferred amino acids at positions 10, 14, 19 and 30, one of the 20 proteinaceous L-amino acids at position 2.

Preferred exendin analogs are exendin analogs which have a substitution at position 3 or 14, particularly preferably at position 2, very preferably comprising only proteinaceous amino acids.

In addition to the newly shortened and stabilized exendin-3 and exendin-4 analogs, the present invention also relates to analogs in amino acids in which the analogs correspond to amino acids in the linked analogs, optionally supplemented with corresponding amino acids that are not present in natural exendin peptides. And methods for preparing analogs by solid phase synthesis from protected amino acids.

Glycine at position 30 of the exendin-3 or exendin-4 configuration is substituted with other proteinaceous or nonproteinaceous amino acids to separate the amino terminal protecting groups and to prevent diketopiperazine formation during synthesis.

Exendin- (1-30) analogs and fragments are advantageous over exendin-1- (1-39) because the short arrangement of these analogs allows for simpler synthesis with high yields.

The abbreviations and definitions of amino acids are as recommended in Pure Appl. Chem. 31, 639-45 (1972) and 40, 277-90 (1974) and are consistent with the PCT rules (WIPO Regulation 23; Recommendation). for the Presentation of Nucleotide and Amino Acid Sequence in Patent Application and in Published Patent Documents). Date and three-letter symbols are as follows.

Amino acid abbreviation

Amino Acid Triangle Symbol

Alanine Ala A

Arginine Arg R

Asparagine Asn N

Aspartic Acid Asp D

Cysteine Cys C

Glutamine Gln Q

Glutamic Acid Glu E

Glycine Gly G

Histidine His H

Isoleucine Ile I

Leucine Leu L

Lysine Lys K

Methionine Met M

Phenylalanine Phe F

Proline Pro P

Serine Ser S

Threonine Thr T

Tryptophan Trp W

Tyrosine Tyr Y

Valine Val V

Other Amino Acids Xaa X

The symbol represents L-amino acid unless specified as D- or D, L-. D-amino acids are represented by small letters in the date symbols. Certain natural or unnatural amino acids are not chiral and are, for example, glycine. In the designation, the N-terminus of all peptides is on the left and the C-terminus is on the right.

Examples of nonproteinaceous amino acids are listed in the table below with their abbreviations.

β-alanine β-Ala

o-aminobenzoic acid Oab

m-aminobenzoic acid Mab

p-aminobenzoic acid Pab

m-aminomethylbenzoic acid Amb

ω-Aminohexanoic acid Ahx

ω-aminoheptanoic acid Ahp

ω-aminooctanoic acid Aoc

ω-Aminodecanoic acid Ade

ω-Aminotetradecanoic acid Atd

Citrulline Cit

Cyclohexylalanine Cha

α, γ-diaminobutyric acid Dab

α, β-diaminobutyric acid Dap

Methionine sulfoxide Met (O)

C ^α -methyl-alanine Aib

N-methyl-glycine (sarcosine) Sar

Naphthylalanine Nal

Norleucine Nle

Ornithine Orn

1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid Tic

All amino acids can be divided into the following three main regions depending on their physical-chemical properties.

Acidic: Amino acids release protons in aqueous solutions and physiological pH, eventually becoming negatively charged.

Basic: Amino acids acquire protons in aqueous solutions and physiological pH, eventually having a positive charge.

Neutral: Amino acids have an uncharged state in aqueous solution and physiological pH.

The definition of “having a positive / negative charge” or “non-charged state” means that a significant number of statistical averages (at least 25%) of the amino acid classes are in the above-mentioned state.

In addition to the 20 so-called proteinaceous amino acids whose introduction into proteins is regulated by genetic code, nonproteinaceous amino acids can also be introduced into the peptide sequence by the described synthesis. The proteinaceous amino acids and the classification into the three regions described above are listed in Table 1. Nonproteinaceous amino acids are not genetically encoded. Examples of nonproteinaceous amino acids and their classification as acidic, basic or neutral amino acids are listed in Table 1.

Proteinaceous Nonproteinaceous acid Asp, Glu Basic Arg, His, Lys Dab, Dap, Orn neutrality Ala, Asn, Cys, Gln, Gly, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val β-Ala, Aib, Cit, Cha, Oab, Mab, Pab, Amb, Ahx, Ahp, Aoc, Ade, Atd, Nal, Nle, Sar, Tic

Exendin analogs that are a subject of the present invention have useful therapeutic properties. Since they promote the supply of insulin from the endocrine pancreas, they increase the biosynthesis of insulin and promote glucose consumption in the peripheral. Since this effect is only observed when blood glucose levels increase simultaneously, hypoglycemia after its administration is not predicted. Exendin analogs also induce a decrease in your life because they inhibit glucagon secretion from the endocrine pancreas. Exendin analogs in non-insulin dependent diabetes mellitus (NIDDM) drive markedly improved metabolic conditions. In particular, glucose uptake in muscle and adipose tissue increased independently of insulin secretion. It is also reasonable to administer exendin analogs to insulin dependent diabetes due to glucagon secretion inhibitory effects. Compared to glucagon-like peptide 1 (GLP-1) and known exendin-3 and exendin-4 configurations, the exendin analogs of the present invention have surprisingly very high potency in a variety of test systems, resulting in GLP-1, exen It is more suitable for therapeutic applications compared to the Dean-3 and Exendin-4 configurations. The advantages of the new exendin analogs are in particular: high stability against degradation and metabolism, longer duration of action, efficacy at small doses. Particular preference is given to analogs based on exendin-3 that have utility in particular over long periods of time or especially in small doses.

Synthesis of solid and liquid phases is a common method in peptide synthesis. When optimizing the method for synthesizing a particular protein, taking into account the purity and yield of the crude product, the method variables and the materials used, e.g. the support, the reagents which cause the reactors to react, the material which masks the groups which should not react, or Reagents capable of separating the shielding material must be tailored to the product to be synthesized, the intermediate to be synthesized and the starting material. This application is not a simple matter, given the interdependence of many method variables.

Pharmaceutical preparations comprising the peptides according to the invention which comprise the active substances in addition to or in addition to conventional auxiliaries and additives are preferably administered parenterally (subcutaneously, intramuscularly or intravenously). However, as other general dosage forms, oral, anal, buccal (including sublingual), lung, transdermal, iontophoretic, pant and nasal administration may be considered. The drug has the ability to regulate insulin and promotes an effective direction to lower blood sugar levels. The drug is advantageously used when the blood glucose level is between 20 and 50 pmol / l. This requires an injection rate of 0.4-1.2 pmol / kg / min. For subcutaneous or buccal administration, an amount of material of 5-500 nmol is required, depending on the galenus form and the desired duration.

The exendin analogs according to the invention and their pharmaceutically acceptable salts are preferably stored as sterile lyophilisates and mixed in a suitable isotonic solution prior to administration. Analogs may be injected, injected or optionally absorbed through the mucosa in this form. Conventional isotonic systems may be employed as solvents suitable for injection and injection containing common additives for injection solutions such as stabilizers and solution agents. Any isotonic solution made from physiological saline solution or buffer is preferred in this case.

Additives are, for example, to adjust viscosity with tartaric acid or citrate buffers, ethanol, complexing agents (such as ethylene diaminetetraacetic acid and non-toxic salts thereof), high molecular weight polymers (such as liquid polyethylene oxide). The liquid carrier material for the injection solution should be sterile and is preferably filled in ampoules. Examples of solid carrier materials include starch, lactose, mannitol, methyl cellulose, talc, highly dispersed silicic acid, high molecular weight fatty acids (such as stearic acid), gelatin, agar-agar, calcium phosphate, magnesium stearate, animal or vegetable oils Solid high molecular weight polymers (such as polyethylene glycol); if desired, suitable for oral dosage formulations including flavors and sweeteners. Nasal administration may include a surfactant to increase absorption through the nasal mucosa. Examples are choline acid, taurocholine acid, chenodeoxycholine acid, glycolic acid, dihydrocholine acid, dioxycholine acid and cyclodextrins.

Daily dosages range from 150-500 nmol. The determination of bioactivity is based on measuring glucagon-like peptide-1, exendin-3 or exendin-4 for international standard preparations according to conventional assays for glucagon-like peptide-1.

Exendin analogs according to the present invention are known from known literature (for example, JM Steward and JD Young "Solid Phase Peptide Synthesis", 2nd Ed., Pierce Chemical Co., Rockford, Illinois (1984) and J. Meienhofer Hormonal Proteins and Peptides, Vol. 2 Academic Press, New York (1973): As for liquid phase synthesis, E. Schroder and K. Lubke "The Peptides", Vol. 1, Academic Press, New York (1965)). It may be prepared according to a conventional peptide synthesis method as described.

General Method of Peptide Synthesis

Generally, for peptide synthesis, protected amino acids are added to the growing peptide chain. All reactive groups, amino groups and carboxyl groups of the first amino acid side chain are protected. This protected amino acid can be bound to an inert support or used in solution. The next amino acid of the peptide sequence is added to the first amino acid after it is adequately protected under the desired conditions to form amide bonds. When all amino acids are combined in the correct sequence, the protecting group and the support phase are separated. The crude polypeptide thus obtained is reprecipitated and preferably purified by chromatography to form the final product. A preferred method for synthesizing analogs of physiologically active polypeptides with less than 40 amino acids consists of solid phase peptide synthesis. In this process, the α-amino functional group (N ^α ) and any reactive side chains are protected with acid-labile groups or base-labile groups. The protecting group used should be stable under the conditions for linking the amide bonds, but should also be readily cleavable without damaging the polypeptide chains formed. Suitable protecting groups for α-amino functional groups include, but are not limited to, the following groups: t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Z), o-chlorobenzyloxycarbonyl, non Phenylisopropyloxycarbonyl, tert.-amyloxycarbonyl (Amoc), α, α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrosulphenyl, 2-cyano-t-butoxy Carbonyl, 9-fluorenylmethoxycarbonyl (Fmoc), 1- (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene) ethyl (Dde) and the like. 9-fluorenylmethoxycarbonyl (Fmoc) is preferably used as the N ^α -protecting group.

Suitable side chain protecting groups include, but are not limited to, the following groups: acetyl, allyl (All), allyloxycarbonyl (Alloc), benzyl (Bzl), benzyloxycarbonyl (Z), t-butyloxy Carbonyl (Boc), benzyloxymethyl (Bom), o-bromobenzyloxycarbonyl, t-butyl (t-Bu), t-butyldimethylsilyl, 2-chlorobenzyl, 2-chlorobenzyloxycarbonyl ( 2-CIZ), 2,6-dichlorobenzyl, cyclohexyl, cyclopentyl, 1- (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene) ethyl (Dde), isopropyl, 4 -Methoxy-2,3,6-trimethylbenzylsulfonyl (Mtr), 2,3,5,7,8-pentamethylchloman-6-sulfonyl (Pmc), pivalyl, tetrahydropyran-2- 1, Tosyl, 2,4,6-trimethoxybenzyl, trimethylsilyl and trityl (Trt).

In solid phase synthesis, the C-terminal amino acid is initially linked to a suitable support material. Suitable support materials are those which are inert to the reagents and reaction conditions for the stepwise condensation and cleavage reactions and do not dissolve in the reaction medium used. Examples of commercially available support materials include styrene / divinylbenzene copolymers modified with reactor and / or polyethylene glycol and chloromethylated styrene / divinylbenzene copolymers, hydroxymethylated or aminomethylated styrene / divinylbenzene copolymers. Etc. can be mentioned. Polystyrene derivatized with 4-benzyloxybenzyl alcohol (Wang-anchor (Wang, SS 1973)) or 2-chlorotrityl chloride (Barlos, K. et al. 1989) for preparing peptide acids (1%)-divinylbenzene or tentagel (Rapp Polymere, Tubingen) is preferably used. In the case of peptide amides, 5- (4'-aminomethyl) -3 ', 5'-dimethoxyphenoxy) valeric acid (PAL-anchor) (Albericio, F. et al., 1987) or p- ( Preference is given to polystyrene (1%) divinylbenzene or tentagel derivatized with 2,4-dimethoxyphenylaminomethyl) phenoxy group (link-amide anchor; Rink-Amid anchor (Rink, H. 1987)).

The linkage to the polymeric support is C-terminal Fmoc in ethanol, acetonitrile, N, N-dimethylformamide (DMF), dichloromethane, tetrahydrofuran, N-methylpyrrolidone or similar solvents, preferably in DMF. -Protected amino acids can be achieved by adding a support material and an activator to react at room temperature or elevated temperature, for example from 40 ° C. to 60 ° C., preferably from room temperature for 2 to 72 hours, preferably about 2 × 2 hours. have.

Coupling of N ^α protected amino acids, preferably Fmoc amino acids, with PAL, king or link anchors, for example, is preferably 1-hydroxybenzotriazole or 1-hydroxy supplemented with TBTU with HOBt added In the presence or absence of hydroxy-7-azabenzotriazole, for example, a base such as diisopropylethylamine (DIEA), triethylamine or N-methylmorpholine, preferably diisopropylethylamine, is added About 10 ° C. to 50 ° C., under 1.5 times to 3 times excess, preferably 2 times excess amino and coupling agent, for 2 to 72 hours, preferably 3 hours, with or without addition Preferably at a temperature of 25 ° C. in a solvent such as dimethylformamide, N-methylpyrrolidone or dichloromethane, preferably dimethylformamide, N, N′-dicyclohexylcarbodiimide (DCC), N, N'-diisopropylcarbo Imide (DIC) or other carbodiimide, 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) or other uronium salt, o Couples such as acylurea, benzotriazol-1-yl-trispyrrolidinophosphonium hexafluorophosphate (PyBOP) or other phosphonium salts, N-hydroxysuccinimides, other N-hydroxyimides or oximes It can be carried out under the aid of a ring agent. Instead of coupling agents, active esters (eg pentafluorophenyl, p-nitrophenyl, etc.), symmetric anhydrides of N ^α -Fmoc-amino acids, acid chlorides or acid fluorides thereof may also be used under the conditions described above.

The N ^α -protected amino acid, preferably Fmoc amino acid, is preferably coupled with the 2-chlorotrityl resin in a dichloromethane added with DIEA for a reaction time of 10 to 120 minutes, preferably 20 minutes, but the solvent and It is not limited to the use of such bases.

Continuous coupling of protected amino acids can be carried out according to conventional methods for peptide synthesis, typically automated peptide synthesizers. N ^α -Fmoc protecting group of amino acid coupled to the solid phase for 5-20 minutes with piperidine (10% to 50%) in dimethylformamide, preferably 2 × 2 minutes with 50% piperidine in DMF and DMF After cleavage by 1 × 5 min of treatment with 20% piperidine in water, 3 to 10 times excess, preferably 10 times excess, the next protected amino acid is dichloromethane, DMF or a mixture of two solvents, preferably DMF Coupling to the previous amino acid at a temperature of about 10 ° C. to 50 ° C., preferably 25 ° C., in an inert, non-aqueous, polar solvent such as; Reagents already mentioned for coupling the first N ^α -Fmoc amino acid to the PAL, king or link anchor are suitable as coupling agents. Active esters of protected amino acids, their chlorides or fluorides or symmetric anhydrides can also be used as substitutes.

In the final step of solid phase synthesis, the peptide is cleaved from the support material while simultaneously cleaving side chain protecting groups. Cleavage is preferably 5% -20% v / v scavenger, preferably dimethylsulfide, ethylmethylsulfide, thioanisole, thicresol, m-cresol, anisole ethanedithiol, phenol or water 15% v / v dimethylsulfide / ethanedithiol / m-cresol 1: 1: 1 may be added with other strongly acidic media or trifluoroacetic acid for 0.5 to 3 hours, preferably 2 hours. Peptides with fully protected side chains are obtained by cleaving the 2-chlorotrityl anchor with glacial acetic acid / trifluoroethanol / dichloromethane 2: 2: 6. Protected peptides can be purified by silica gel chromatography. If the peptide is linked to the solid phase via a royal anchor and it is desired to obtain a C-terminal alkylamideized peptide, cleavage can be effected by aminolysis with alkylamines or fluoroalkylamines. Aminolysis is carried out at a temperature of about −10 ° C. to 50 ° C., preferably at a temperature of about 25 ° C. for a reaction time of about 12 to 24 hours, preferably about 18 hours. The peptide can also be cleaved from the support, for example by re-esterification with methanol.

The acidic solution obtained is admixed with 3 to 20 times the amount of cold ether or n-hexane, preferably 10 times the excess of diethyl ether to precipitate the peptide, thereby leaving the scavengers and cleaved protecting groups remaining in the ether. Separate. Purification can be performed further by reprecipitating the peptide several times with glacial acetic acid. The obtained precipitate is collected in water or tert-butanol or a mixture of two solvents, preferably in a 1: 1 mixture of tert-butanol / water and freeze-dried.

The obtained peptides can be purified in part or all by the following chromatographic methods: ion exchange chromatography on weakly basic resins in the form of acetates; Hydrophobic adsorption chromatography on non-derivatized polystyrene / divinylbenzene copolymers (eg, Amberlite XAD); Adsorption chromatography on silica gel; Ion exchange chromatography on carboxymethyl cellulose, for example; For example, partition chromatography on Sephadex G-25; Countercurrent chromatography; Or high pressure liquid chromatography (HPLC), in particular reverse phase HPLC on octyl or octadecylsilyl silica (ODS).

In summary, the present invention relates to a method for preparing a polypeptide and a pharmaceutically usable salt thereof. The above method of inducing physiologically active shortened analogs and homologs of exendin-3 or exendin-4 having the above-mentioned preferred chain lengths and modifications can be achieved by sequentially condensing protected amino acids on suitable support materials, supports and A method for cleaving a protecting group and a method for purifying the obtained peptides.

Amino acid analysis is performed with amino acid analyzer 420 A (Applied Biosystems Company; Weiterstadt). 50 to 1000 pmol of the sample to be analyzed is added to a sample carrier in a 10 to 40 μl solution and then completely hydrolyzed at 160 ° C. for 90 minutes with 6 N hydrochloric acid in the gas phase, derivatized with phenylisothiocyanate and microbore ) On-line analysis by HPLC. Mass spectroscopic investigations are performed with an API III triple-quadruple mass spectrometer (SCIEX; Thronhill, Canada) equipped with an ion source of ion spray.

Protected amino acid derivatives can be obtained for example from Novabiochem GmbH (Bad Soden).

The following examples illustrate exemplary parts of the inventive concept and do not limit the subject matter of the invention.

Example 1

HGEGTFTSDLSKQ-Nle-EEEAVRLFIEWLKNGR-NH ₂ (SEQ ID NO: 3)

[NLe ¹⁴ , Arg ³⁰ ] -exendin-4- (1-30) -NH ₂

Example 1 was prepared using 5-((4'-aminomethyl) -3 ', 5'-dimethoxyphenoxy) valeranyl-alanyl-aminomethyl using MultiSyn Tech's (Bochum) multi-automated peptide synthesizer SyRo II Synthesized in -0.02 mmol batch by solid phase method on -polystyrene (1%) divinylbenzene (load: 0.5 mmol / g). The α-amino functional group of the amino acid was protected with 9-fluorenylmethoxycarbonyl (Fmoc). The side chain protecting groups are assigned to t-butyl (tBu) for Asp, Glu, Ser and Thr, trityl (Trt) for Asn, Gln and His, t-butyloxycarbonyl (Boc) for Lys and Trp, and Arg. For 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc). Protected amino acids were continuously coupled in 10-fold excess using N, N-diisopropylcarbodiimide / 1-hydroxybenzo-triazole as activator and double coupling twice for 40 minutes. Peptides are separated from the polymer support and at the same time trifluoroacetic acid (85%) in the presence of 15% ethanedithiol / dimethylsulfide / m-cresol (1: 1: 1 v / v / v) for 120 minutes at room temperature. The protecting group was cut in the middle. The peptide was then precipitated with anhydrous diethyl ether and then washed several times with anhydrous diethyl ether to completely remove the thiol. The precipitate was freeze-dried from water / tert-butanol (1: 1) to give 62 mg of the peptide. Zopeptide was purified within 30 minutes by reverse phase HPLC using a gradient of 37% to 42% acetonitrile / 0.9% TFA. The eluate was evaporated and lyophilized to yield 29 mg of a white solid having a purity of at least 97%.

Amino acid analysis: Ala 1.08 (1); Asx 1.91 (2); Glx 6.10 (6); Phe 1.78 (2); Gly 3.10 (3); His 1.00 (1); Ile 0.88 (1); Lys 2.02 (2); Leu 3.24 (3); Nle 1.10 (1); Arg 1.98 (2); Ser 2.04 (2); Thr 1.99 (2); Val 0.91 (1); Trp O..87 (1)

ESI-MS: 3488.2

Example 2

HGEGTFTSDLSKQ-Nle-EEEAVRLFIEWLKNGY-NH ₂ (SEQ ID NO: 4)

[Nle ¹⁴ , Tyr ³⁰ ] -exendin-4- (1-30) -NH ₂

Example 2 is a link-amide anchor (4- (2 ', 4'-dimethoxyphenyl-aminomethyl) -phenoxy group) using a multiautomated peptide synthesizer SyRo II from MultiSynTech (Bochum) (load: 0.18 mmol / g) was synthesized in a 0.0076 mmol batch by a solid phase method on Tentagel ™ (Rapp Polymers, Tubingen) derivatized with g). The protected amino acids used were similar to Example 1. Protected amino acids were coupled in a single 8-fold excess in series with a single coupling with stirring at 40 ° C. for 40 minutes. 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyl-uronium tetrafluoroborate (TBTU) / 1-hydroxybenzotriazole by addition of diisopropylethylamine Was used as activator. The peptide was cleaved and purified similarly to Example 1. 18.1 mg of a white solid having a purity of more than 95% was obtained.

Amino acid analysis: Ala 1.03 (1); Asx 1.90 (2); Glx 6.24 (6); Phe 1.94 (2); Gly 3.12 (3); His 1.02 (1); Ile 1.09 (1); Lys 2.01 (2); Leu 3.06 (3); Nle 1.08 (1); Arg 0.97 (1); Ser 1.98 (2); Thr 1.80 (2); Val 0.93 (1); Trp 1.01 (1); Tyr 0.90 (1).

ESI-MS: 3494.8

Example 3

HSDGTFTSDLSKQ-Nle-EEEAVRLFIEWLKNGR-NH ₂ (SEQ ID NO: 5)

[Nle ¹⁴ , Arg ³⁰ ] -exendin-3- (1-30) -NH ₂

Example 3 was synthesized similar to the method described in Example 2. 17.6 mg of a white solid having a purity of at least 99% was obtained.

Amino acid analysis: Ala 0.99 (1); Asx 2.98 (3); Glx 5.16 (5); Phe 2.08 (2); Gly 2.16 (2); His 0.95 (1); Ile 1.03 (1); Lys 2.04 (2); Leu 2.91 (3); Nle 1.05 (1); Arg 1.04 (1); Ser 3.00 (3); Thr 2.05 (2); Val 1.01 (1); Trp 1.18 (1); Tyr 0.98 (1).

ESI-MS: 3504.4

Example 4

HGEGTFTSDLSKQMEEEAVRLFIEWLKNGR-NH ₂ (SEQ ID NO: 6)

[Arg 3 ^O ] -Exendin-4- (1-30) -NH ₂

Example 4 was synthesized similar to the method described in Example 1. 17.9 mg of a white solid having a purity of at least 96% was obtained.

Amino acid analysis: Ala 0.96 (1); Asx 2.01 (2); Glx 6.00 (6); Phe 1.80 (2); Gly 3.21 (3); His 0.96 (1); Ile 1.07 (1); Lys l.92 (2); Leu 2.98 (3); Met 1.06 (1); Arg 1.90 (2); Ser 1.91 (2); Thr 2.09 (2); Val 0.97 (1); Trp 0.84 (1).

ESI-MS: 3508.4

Example 5

GEGTFTSDLSKQ-Nle-EEEAVRLFIEWLKNGR-NH ₂ (SEQ ID NO: 7)

[Nle ¹⁴ , Arg ³⁰ ] -exendin-4- (2-30) -NH ₂

Example 5 was synthesized similar to the method described in Example 2. 13.2 mg of a white solid having a purity of at least 97% was obtained.

Amino acid analysis: Ala 1.04 (1); Asx 1.98 (2); Glx 6.08 (6); Phe 1.86 (2); Gly 2.91 (3); Ile 0.96 (1); Lys 1.84 (2); Leu 2.98 (3); Nle 1.04 (1); Arg 1.90 (2); Ser 1.94 (2); Thr 1.92 (2); Val 0.96 (1); Trp 0.85 (1).

ESI-MS: 3350.8

Example 6

HGEGTFTSDLSKQMEEEAVRAFIEWLKNGR-NH ₂ (SEQ ID NO: 8)

[Ala ²¹ , Arg ³⁰ ] -exendin-4- (1-30) -NH ₂

Example 6 was synthesized similar to the method described in Example 1. 11.1 mg of a white solid having a purity of at least 95% was obtained.

Amino acid analysis: Ala 2.08 (2); Asx 1.93 (2); Glx 6.07 (6); Phe 1.74 (2); Gly 2.97 (3); His 0.98 (1); Ile 0.87 (1); Lys 2.15 (2); Leu 2.02 (2); Met 0.96 (1); Arg 2.13 (2); Ser 1.87 (2); Thr 2.07 (2); Val 1.04 (1); Trp 0.87 (1).

ESI-MS: 3466.3

Example 7

HGEGTFTSDLSKQMEEEAVRLFIEWLKAGR-NH ₂ (SEQ ID NO: 9)

[Ala ²⁸ , Arg ³⁰ ] -exendin-4- (1-30) -NH ₂

Example 7 was synthesized similar to the method described in Example 1. 15.0 mg of a white solid having a purity of at least 97% was obtained.

Amino acid analysis: Ala 1.98 (2); Asx O. 98 (1); Glx 6.22 (6); Phe 1.92 (2); Gly 3.03 (3); His 0.99 (1); Ile 1.03 (1); Lys 2.05 (2); Leu 3.03 (3); Met 0.96 (1); Arg 1.84 (2); Ser 1.98 (2); Thr 2.09 (2); Val 1.01 (1); Trp 0.72 (1).

ESI-MS: 3465.4

Example 8

HGEGTFTSDLSKQMEEEAVRAFIEWLKAGR-NH ₂ (SEQ ID NO: 10)

[Ala ^21,28 , Arg ³⁰ ] ^-exendin -4- (1-30) -NH ₂

Example 8 was synthesized similar to the method described in Example 1. 18.4 mg of a white solid having a purity of at least 95% was obtained.

Amino acid analysis: Ala 3.12 (3); Asx 0.99 (1); Glx 6.04 (6); Phe 1.80 (2); Gly 3.OO (3); His 0.96 (1); Ile 1.02 (1); Lys 1.84 (2); Leu 1.97 (2); Met 0.98 (1); Arg 2.03 (2); Ser 1.91 (2); Thr 1.88 (2); Val 0.99 (1); Trp 0.99 (1).

ESI-MS: 3423.3

Example 9

In a similar manner the following high purity exendin derivatives could be prepared.

(Ex-4 = Exendin-4, Ex-3 = Exendin-3)

Exendin-derivative sequence number sequence

The following example shows derivatization of aminomethylpolystyrene (1%) divinylbenzene with link amide anchor (4- (2 ', 4'-dimethoxyphenyl-aminomethyl) -phenoxy group) (load: 0.5 mmol / g) Was synthesized in 0.02 mmol batch by solid phase method on RAM resin (trade name) (Rapp Polymers, Tubingen). The synthesis was carried out using a multiautomated peptide synthesizer SyRo II from MultiSyn Tech (Bochum). The protected amino acids used were similar to those of Example 1. Protected amino acids were coupled in 5-fold excess continuously by single coupling at 40 ° C. for 40 minutes with stirring. Diisopropylethylamine was added to use 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) as the activator. Cleavage and purification of the peptide was performed similarly to Example 1. Yield, purity and assay data of the synthesized peptides described above are listed in the following tables.

TABLE 1

Sequence Number Yield [mg] Purity [%] ESI-MS

Example 10 Pharmacological Data

Peptide Metabolism in Preparation of Etopeptidase or in Preparation of Kidney Microvilli

background

The group of ecopeptidases contributes to the post-secretory metabolism of peptide hormones. These enzymes bind to the cytoplasmic membranes of various cell types. Their active site is towards the extracellular space. In addition, these enzymes are present at high concentrations in the chain membranes of the proximal tubules. Thus, the renal chain microvilli (BBM) are a suitable source for appropriate ecopeptidase and can be used as in vitro tests for the metabolic stability of synthetic peptides. Alternatively, it can be used for the preparation of ecopeptidase. Dipeptidyl peptidase IV as well as human neutral endopeptidase 24.11 are used as examples because GLP-1 is the substrate of all of the ectopeptidases.

Preparation of Chain Natural Microvilli

Cytoplasmic inhibition cleavage using fractional centrifugation to isolate microvilli of rat and porcine kidney medulla (Booth and Kenny (1975)). To evaluate purity and membrane yield, four chain ectopic peptidases are detected by fluorometry and other marker enzymes are measured by colorimetry.

Ectopeptidase Agents

Purified human neutral endopeptidase 24.11 is obtained in recombinant form (Genentech, San Francisco, USA) and dipeptidyl peptidase IV is obtained by isolating from the human placenta (Calbiochem (Bad Soden)).

Incubation Protocol

The microvilli (0.5-1 μg protein) or each ectopeptidase preparation (60-300 ng) was prepared using 10 μg of peptide (about 3 nmol) in 100 μl of HEPES buffer (50 mM, pH 7.4) containing 50 mM NaCl. Incubate with). The reaction is terminated by boiling for a predetermined time (up to 1 hour). The sample is then centrifuged (10,000 × g), diluted with 150 μl of 0.1% TFA and analyzed by reverse phase (RP) HPLC. Each sample is measured twice.

HPLC analysis

A system with the following components is used for HPLC analysis: 2248 low pressure pump (Pharmacia-LKB, Freiburg), WISP 10B autoinjector (Millipore-Waters, Eschborn), UV detector SP-4 (Gynkotec, Berlin), low pressure mixing system ( Pharmacia-LKB, Freiburg) and Program Manager Software Controller (Pharmacia-LKB, Freiburg). Transfer solvent A: 0.1% trifluoroacetic acid (TFA) and B: acetonitrile: water: TFA (70: 29: 0.1), using a binary gradient, Lichrospher C-8 (5 μ, 4 × 124 mm) (Merk, Daemstadt)), the separation is carried out. After injecting 244 μl of sample solution on a column equilibrated with moving solvent A, the incubation product is eluted for 80 minutes with 0% to 80% of B in the first gradient, and UV absorption is measured at 215 nm.

Calculation of proteolysis rate

Measured twice during each incubation period of each peptide, the average peak height of the substrate peak is plotted as a function of time. Using GLP-1 as an example, it can be seen that the peak height in the sample solution is linearly proportional to the amount of peptide. In addition, a linear decrease in peak height over time is observed during the first hour of incubation with microvilli or peptidase. Thus, the proteolysis rate is measured from the decrease in the height of the substrate peak and is expressed in [μmol substrate / mg protein / min].

Degradation Stability of Exendin Homolog

Incubation with human neutral endopeptidase 24.11b

[Nle ¹⁴ , Arg ³⁰ ] -Exendin-4- (1-30) -NH ₂ (SEQ ID No. 3) is incubated with the above-mentioned neutral endopeptidase 24.11 and the degradation rate is measured. GLP-1- (7-36) -NH ₂ is used as a control. The results are shown in Table 3 below.

Decomposition Rate [mM / 100ng / ml NEP24.11 / min] GLP1- (7-36) -NH ₂ 0.0586 [Nle ¹⁴ , Arg ³⁰ ] -Ex-4- (1-30) -NH ₂ Example 1 0.0083

Incubation with Dipeptidyl Peptidase IV

As mentioned above, the peptides listed in Table 4 are incubated with dipeptidyl peptidase IV (DDP-IV). When GLP-1- (7-36) -NH ₂ is 50% hydrolyzed, each incubation is terminated. Substrate peaks of each peptide are collected from the rpHPLC test and examined by mass spectrometry to exclude cleaved products.

Homologue Seq.ID Substrate for DDP-IV [Lys ² , Nle ¹⁴ , Arg ³⁰ ] -Ex-4- (1-30) -NH ₂ 78 No proteolysis GLP1- (7-36) -NH ₂ 50% proteolysis

Incubation with a suppurative microvilli

Table 5 shows the proteolysis rates calculated after incubation with the natural microvilli (BBM) according to the above protocol. GLP1- (7-36) -NH ₂ is used as a control.

Homologue Seq.ID Degradation rate [ng peptide / min / mg BBM] GLP1- (7-36) -NH ₂ 880,00 [Lys ² , NlE ¹⁴ , Arg ³⁰ ] -Ex-3- (1-30) -NH ₂ 86 2.05

Insulin secretion by isolated pancreatic islet cells

Organ removal

Anesthetized (0.3-0.5 ml nembutal / isophysiological saline 1: 4, intraperitoneal) mice are opened by midline incisions to fix the incision side, peritoneum, and incision in the rib cage along the diaphragm. All organs are inflated and stained red by injecting a neutral red solution into the left cavity. Carefully remove the pancreas along the stomach and duodenum to the mesentery. The pancreas is placed in an ice-cold Petri dish in Hank Balance Salt Solution (HBBS) until a few minutes of digestion, and several drops of neutral red solution are dropped.

Island manufacturing

Tap the two pancreas with cellulose and place in the tube. 5 ml of freshly prepared collagenase solution (C. histolyticum 0.74 U / mg, Serva, 2 mg / ml in HBBS / water 1: 9, pH 7.4) was added and shaken at 18 ° C. at 18 ° C. Incubate for minutes. Then, it is centrifuged at 1000 rpm for 1 minute. Discard the supernatant. In the second digestion step, 5 ml of collagenase solution (1 mg / ml) is incubated for 4 minutes, and shaken to precipitate undigested tissue. Discard the supernatant and repeat the entire procedure 4-5 times. The supernatant is then centrifuged at 1000 rpm for 1 minute and the collagenase solution is discarded. The remaining pellets are shaken with ice cold HBBS and allowed to settle on ice for about 10 minutes. This washing procedure is repeated three more times. The pink-stained islets under stereoscopic magnification were collected from the washed pellets and cultured medium (RPMI 1640 (Gibco) 100 ml, glutamine 1 ml, penicillin 1 ml, Cibrobay antibiotic (Bayer), calf fetal serum 10 ml, 1M 2 ml of Hepes buffer). To obtain as pure a culture as possible, collect islets 2-3 times and transfer to fresh culture medium.

Stimulation of the island

Islet cells collected from the culture medium were prepared in stimulation buffer (NaCl 118 mM, NaH ₂ PO ₄ 0.2 mM, MgCl ₂ 0.565 mM, CaCl ₂ 1.25 mM, KCl 4.7 mM, Hepes 10 mM, in an amount of 10 islets per Eppendorf tube). Aliquot into an Eppendorf tube containing 200 ml of BSA 1%, 3.3 mM glucose; pH 7.4) and place in the incubator at 37 ° C. for 1 hour. The peptide to be tested is then added and filled with 500 ml with stimulation buffer and incubated at 37 ° C. for 1 hour. The islands are centrifuged at 1000 rpm for 1 minute. Insulin-RIA (DPC Biermann, Nauheim) is used to determine the amount of C-peptide in the supernatant. Each test substance is measured in quadruplicate.

Activity of exendin analogs

As described above for isolated islet cells, several exendin analogues are tested for insulin secretion activity. The data is shown in the following table as an example.

After 1 hour in the presence of 10 mM glucose, insulin secretion from isolated isles [mIU / h / 10 islets]:

contrast GLP1- (7-36) -NH ₂ Seq. ID 84 10 mM glucose 30.21 10 ^-7 (10 mM glucose) 53.52 48.94 10 ^-8 (10 mM glucose) 42.78 41.72 10 ^-9 (10 mM glucose) 29.99 38.76 10 ^-10 (10 mM glucose) 35.05

Increased measurement of cytosolic calcium levels in B cells of the endocrine pancreas (clonal B cell line INS-1)

Culture of INS-1-cells (Asfari, M., 1992):

INS-1 cells were treated with FCS 10%, HEPES buffer (pH 7.4) 10 mM, L-glutamine 2 mM, penicillin 100 i.U. Incubate in 37 ° C., 95% air and 5% carbon dioxide in RPMI 1640 medium containing / ml, streptomycin 100 μg / ml, sodium pyruvate 1 mM, and 50 μM 2-mercaptoethanol. After incubation in a plastic cell culture plate for 6-8 days, the fusion cells are rinsed once with PBS (phosphate buffered saline) and then incubated for 4 minutes at 37 ° C. with isotonic saline containing 0.025% trypsin and 0.27 mM EDTA for 4 minutes. Isolate.

Preparation of Cells for Calcium Measurement:

The separated cells are resuspended in Spinner medium (culture medium as above except 5% of FCS and 25 mM HEPES) and incubated for 2 hours and 30 minutes in a Spinner bottle equipped with a stirring bar. The medium is then removed by centrifugation and the cells are resuspended in Spinner medium. Subsequently, incubate for 30 minutes at 37 DEG C with 2 [mu] M Fura-2 / acetoxymethyl ester under the same conditions as described above. Spinner medium is washed from the cells to measure the Fura load of the cells (room temperature). The cells are then resuspended in Spinner medium at room temperature (2 × 10 ⁷ cells / ml). The cells are then removed from the suspension for calcium measurement.

Measurement of cytosolic calcium concentrations:

NaCl 136 mM, KCl 4.8 mM, CaCl ₂ 2 mM, MgSO ₄ 1.2 mM, KH ₂ PO ₄ 1.2 mM, NaHCO ₃ 5 mM, Glucose 10 mM, Sulpinpyrazone 250 μM (prevents the entry of Fur-2 into the medium ), And modified Krebs-Ringer buffer (KRBH) containing 25 mM HEPES buffer (adjusted to pH 7.4 with NaOH) at 37 ° C. Cell concentration shall be 1-2x10 <^6> / ml. 1.5 ml of the cell suspension is measured by stirring with a stir bar at 37 ° C. in a cuvette in a spectrophotometer. The excitation wavelength is 340 nm and emission wavelength is 505 nm. At the end of the measurement, 50 μM of MnCl ₂ was added followed by 100 μm of DTPA (diethylenetriamine pentaacetate) to temporarily quench the fluorescence of the extracellular Fura, thereby reducing the ratio of the extracellular fluorescence indicator to the measured fluorescence. Measure After the addition of DTPA, the entire Fura is first calcium saturated and then switched to the calcium free state to determine the calibration values of F _max (calcium saturated state) and F _min (calcium free) for each measurement. To this end, 0.1% of Triton X-100 is added to lyse the cells. The dye is saturated with calcium by contacting at high extracellular calcium concentrations. Then, 5 mM EGTA (ethylenebis (oxyethylenenitrile) -tetraacetate) and 20 mM Tris solution are added to convert the dye to the calcium free form.

Cytoplasmic calcium ion concentrations are calculated according to the following formula (R. Tsien et al., Grynkiewicz, G., 1985):

[Ca ²⁺ ] _cyt = ((F-F _min ) / (F _max -F)) x K _D

(F: fluorescence of each measuring point;

K _D : dissociation constant of calcium complex of Fura-2, 225 nM (Grynkiewicz, G., 1985)).

(Before the calculation, compensation for the presence of extracellular Fura is performed. For this purpose, the amount of fluorescence measured by manganese addition (extracellular Fura) is first subtracted from the fluorescence value of the measurement point. Calculate the F _max by 끝 Finally, calculate the correction for F _min To divide the amount of fluorescence measured by the addition of manganese by 2.24, the value of 2.24 is calcium saturated Fura at excitation wavelength 340 nm. Intrinsic mechanical proportionality constant between -2 and the fluorescence value of Fura-2 without calcium (measured with unesterified free Fura-2). The value calibrated in this way is subtracted from F _min .)

A detecting peptides is added as a solution KRBH (10 ^-5 M) of a 1000-fold concentration with no CaCl ₂ and glucose.

Activity of exendin analogs

The biological activity of various exendin analogs is tested as in the calcium assay for INS-1 cells above. The data is shown in Fig. 1 and Table 7 as an example.

SEQ. ID Nr. Concentration of peptide 10 ^-8 M Δ [Ca ²⁺ ] _cyt ± SD (n = 4) 6 [Arg ³⁰ ] -Exendin- (1-30) -NH ₂ 64 ± 8 nM 3 [Nle ¹⁴ , Arg ³⁰ ] -exendin- (1-30)-NH ₂ 63 ± 8 nM 8 [Ala ²¹ , Arg ³⁰ ] -exendin- (1-30)-NH ₂ 61 ± 11 nM 9 [Ala ²⁸ , Arg ³⁰ ] -exendin- (1-30)-NH ₂ 65 ± 15 nM 10 [Ala ^21,28 , Arg ³⁰ ] ^-exendin- (1-30) -NH ₂ 69 ± 30 nM Control: GLP-1- (7-36) amide 65 ± 10 nM

GLP-1- (7-36) -NH on B cells of the endocrine pancreas (clonal B cell line INS-1) ₂ Competition

Culture of INS-1-cells (Asfari, M., 1992)

See measurement of calcium concentration

Competition experiment

The isolated cells were taken and adjusted to Krebs-Ringer buffer (25 mM Tris, 120 mM NaCl, 1.2 mM MgSO ₄ , 5 mM KCl, 1 mM Na-EDTA, 15 mM CH ₃ COONa, pH 7.4, 1% HSA and 0.1% Sucitracin). For the reaction mixture in which 30 ml of peptide to be detected and 20 ml of tracer ( ^125I- GLP1-7-36) -NH ₂ 20,000 rpm) are mixed in the corresponding dilution, 250 ml are removed from the suspension each hour. Subsequently, the cells are incubated at 37 ° C for 30 minutes, centrifuged at 13,000 rpm for 4 minutes, washed three times with buffer, and the radioactivity (γ-reverse direction) attached to the pellets is measured. By culturing with (10 ^-10 ~10 ^-6 M in Krebs-Ringer buffer solution), 10 different dilutions of the peptide to be tested to obtain a competition curve.

Receptor Affinity of Exendin Analogs

The data is shown in Table 8 below as an example. GLP-1- (7-36) -NH ₂ was used as a control.

Seq. ID Peptide Kd _GLP1 ± SD [nM] Kd ± SD [nM] Kd / Kd _GLP1 69 [Nle ¹⁴ , Try ³⁰ ] -Ex3- (1-30) -NH ₂ 1.04 ± 0.05 0.56 ± 0.08 0.5

references

Albericio, F. and Barany, G. (1987) Int. J. Peptide Protein Res. 30 , 206-216.

Asfari, M., Janjic, D., Meda, P., Li, G., Halban, PA and Wollheim, CB (1992) Endocrinology 130 , 167-178.

Barlos, K., Gatos, D., Kapolos, S., Paphotiu, G., Schafer, W., and Wengqing, Y. (1989) Tetrahedron Lett . 30 , 3947-3950.

Booth, and Kenny, (1975) Biochem. J. 142 , 575-581.

Eng, J., and rews, PC, Kleinman, WA, Singh, L., and Raufman, J.-P. (1990) J. Biol. Chem. 265 , 20259-20262.

Eng, J., and rews, PC, Kleinman, WA, Singh, L., Singh, G., and Raufman, J.-P. (1992) J. Biol. Chem. 267 , 7402-7405.

Fehmann, HC, Goke, R., Goke, B., Bachle, R., Wagner, B. and Arnold, R. (1991) Biochem. Biophys. Acta 1091 , 356-63.

Goke, R., Wagner, B., Fehmann, HC and Goke, B. (1993a) Res. Exp. Med. Berl. 193 , 97-103

Goke, R., Fehman, HC, Linn, T., Schmidt, E., Eng, J. and Goke, B. (1993b) J. Biol. Chem. 268, 19650-19655.

Grynkiewicz, G., Poenie, M. and Tsien, RY (1985) J. Biol. Chem. 260 , 3440-3450.

Gutniak, M., orskov, C., Holst, JJ, Ahren, B. and Efendic, S. (1992) N. Engl. J. Med. 326 , 1316-1322.

Komatsu, R., Matsuyama, T., Namba, M., Watanabe, N., Itoh, H., Kono, N., and Tarui, S. (1989) Diabetes 38 , 902-905.

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Nauck, MA, Kleine, N. orskov, C., Holst, JJ, Willms, B. and Creutzfeld, W. (1993a) Diabetclogia 36 , 741-744.

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[Sequence list]

(1) General Information

(i) Applicant:

(A) Name: Boehringer Mannheim Geembeha

(B) Distance: Zanthopper Strasse 116

(C) Municipality: Mannheim

(E) Country: Germany

(F) ZIP Code: DE-68305

(G) Phone: 0621 / 759-4457

(H) FAX: 0621 / 759-4457

(ii) Name of the invention: Exendin analog, preparation method thereof and the same

Pharmaceutical preparations

(iii) number of sequences: 118

(iv) computer readable form

(A) Media Type: Floppy Disk

(B) Computer: IBM PC compatible model

(C) Operating system: PC-DOS / MS-DOS

(D) Software: Patent # 1.0, Edition # 1.30B (EPO)

(2) Information about SEQ ID NO: 1:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence location: 30

(D) Other information: / Product = "Xaa represents all amino acids except Gly"

(xi) Sequence description: SEQ ID NO: 1:

(2) Information about SEQ ID NO: 2:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(xi) Sequence description: SEQ ID NO: 2:

(2) Information about SEQ ID NO: 3:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence description: SEQ ID NO: 3:

(2) Information about SEQ ID NO: 4:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Method of describing sequence: SEQ ID NO: 4:

(2) Information about SEQ ID NO: 5:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence description: SEQ ID NO: 5:

(2) Information about SEQ ID NO: 6:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence description: SEQ ID NO: 6:

(2) Information about SEQ ID NO: 7

(i) the nature of the sequence:

(A) Length of sequence: 29 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence location: 13

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence description: SEQ ID NO: 7:

(2) Information about SEQ ID NO: 8:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence description: SEQ ID NO: 8:

(2) Information about SEQ ID NO: 9:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence description: SEQ ID NO: 9:

(2) Information about SEQ ID NO: 10:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 10

(2) Information about SEQ ID NO: 11:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence description: SEQ ID NO: 11:

(2) Information about SEQ ID NO: 12:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) How to describe the sequence: SEQ ID NO: 12

(2) Information about SEQ ID NO: 13:

(i) the nature of the sequence:

(A) Length of sequence: 27 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 13:

(2) Information about SEQ ID NO: 14:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 14

(2) Information about SEQ ID NO: 15:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 15:

(2) Information about SEQ ID NO: 16:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C)

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Method of describing sequence: SEQ ID NO: 16:

(2) Information about SEQ ID NO: 17:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 17:

(2) Information about SEQ ID NO: 18:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 18

(2) Information about SEQ ID NO: 19:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 19:

(2) Information about SEQ ID NO: 20:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 20

(2) Information about SEQ ID NO: 21:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 21:

(2) Information about SEQ ID NO: 22:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 22:

(2) Information about SEQ ID NO: 23:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 23:

(2) Information about SEQ ID NO: 24:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 24:

(2) Information about SEQ ID NO: 25:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 25

(2) Information about SEQ ID NO: 26:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 26

(2) Information about SEQ ID NO: 27:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 27:

(2) Information about SEQ ID NO: 28:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 28:

(2) Information about SEQ ID NO: 29:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 29

(2) Information about SEQ ID NO: 30:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 30

(2) Information about SEQ ID NO: 31:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 31:

(2) Information about SEQ ID NO: 32:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 32

(2) Information about SEQ ID NO: 33:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) How to describe the sequence: SEQ ID NO: 33:

(2) Information about SEQ ID NO: 34:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 34:

(2) Information about SEQ ID NO: 35:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 35:

(2) Information about SEQ ID NO: 36:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 36:

(2) Information about SEQ ID NO: 37:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Method of describing sequence: SEQ ID NO: 37:

(2) Information about SEQ ID NO: 38:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 38:

(2) Information about SEQ ID NO: 39:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 39

(2) Information about SEQ ID NO: 40:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 40

(2) Information about SEQ ID NO: 41:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 41:

(2) Information about SEQ ID NO: 42:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 42

(2) Information about SEQ ID NO: 43:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) how to describe the sequence: SEQ ID NO: 43:

(2) Information about SEQ ID NO: 44:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 44:

(2) Information about SEQ ID NO: 45:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 45:

(2) Information about SEQ ID NO: 46:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 46

(2) Information about SEQ ID NO: 47:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 47:

(2) Information about SEQ ID NO: 48:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 48:

(2) Information about SEQ ID NO: 49:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 49:

(2) Information about SEQ ID NO: 50:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 50

(2) Information about SEQ ID NO: 51:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 51:

(2) Information about SEQ ID NO: 52:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Method of describing sequence: SEQ ID NO: 52:

(2) Information about SEQ ID NO: 53:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 53:

(2) Information about SEQ ID NO: 54:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 54:

(2) Information about SEQ ID NO: 55:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 55

(2) Information about SEQ ID NO: 56:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 56:

(2) Information about SEQ ID NO: 57:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 57:

(2) Information about SEQ ID NO: 58:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 58

(2) Information about SEQ ID NO: 59:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 59:

(2) Information about SEQ ID NO: 60:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 60

(2) Information about SEQ ID NO: 61:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 61:

(2) Information about SEQ ID NO: 62:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 62

(2) Information about SEQ ID NO: 63:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Method of describing sequence: SEQ ID NO: 63:

(2) Information about SEQ ID NO: 64:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 64

(2) Information about SEQ ID NO: 65:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 65

(2) Information about SEQ ID NO: 66:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 66:

(2) Information about SEQ ID NO: 67:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 67:

(2) Information about SEQ ID NO: 68:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 68

(2) Information about SEQ ID NO: 69:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 69:

(2) Information about SEQ ID NO: 70:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 70:

(2) Information about SEQ ID NO: 71:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 71:

(2) Information about SEQ ID NO: 72:

(i) the nature of the sequence:

(A) Length of sequence: 29 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence location: 13

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 72

(2) Information about SEQ ID NO: 73:

(i) the nature of the sequence:

(A) Length of sequence: 28 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence location: 12

(D) Other information: / product = "Xaa represents Nle"

(xi) Method of describing sequence: SEQ ID NO: 73:

(2) Information about SEQ ID NO: 74:

(i) the nature of the sequence:

(A) Length of sequence: 29 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence location: 13

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 74:

(2) Information about SEQ ID NO: 75:

(i) the nature of the sequence:

(A) Length of sequence: 28 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence location: 12

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 75

(2) Information about SEQ ID NO: 76:

(i) the nature of the sequence:

(A) Length of sequence: 27 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) How to describe the sequence: SEQ ID NO: 76

(2) Information about SEQ ID NO: 77:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 77:

(2) Information about SEQ ID NO: 78:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 78

(2) Information about SEQ ID NO: 79:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 79

(2) Information about SEQ ID NO: 80:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 80

(2) Information about SEQ ID NO: 81:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 81:

(2) Information about SEQ ID NO: 82:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 82:

(2) Information about SEQ ID NO: 83:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Method of describing sequence: SEQ ID NO: 83:

(2) Information about SEQ ID NO: 84:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 84

(2) Information about SEQ ID NO: 85:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 85:

(2) Information about SEQ ID NO: 86:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 86:

(2) Information about SEQ ID NO: 87:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Method of describing sequence: SEQ ID NO: 87:

(2) Information about SEQ ID NO: 88:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 88

(2) Information about SEQ ID NO: 89:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 89

(2) Information about SEQ ID NO: 90:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 90

(2) Information about SEQ ID NO: 91:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 91

(2) Information about SEQ ID NO: 92:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 92

(2) Information about SEQ ID NO: 93:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 93

(2) Information about SEQ ID NO: 94:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 94:

(2) Information about SEQ ID NO: 95:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 95

(2) Information about SEQ ID NO: 96:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(ix) sequence characteristics:

(A) Characteristic symbol: peptide

(B) Presence Location: 14

(D) Other information: / product = "Xaa represents Nle"

(xi) Sequence Description: SEQ ID NO: 96

(2) Information about SEQ ID NO: 97:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 97:

(2) Information about SEQ ID NO: 98:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 98:

(2) Information about SEQ ID NO: 99:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 99:

(2) Information about SEQ ID NO: 100:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 100

(2) Information about SEQ ID NO: 101:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 101:

(2) Information about SEQ ID NO: 102:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 102:

(2) Information about SEQ ID NO: 103:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 103:

(2) Information about SEQ ID NO: 104:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 104:

(2) Information about SEQ ID NO: 105:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Method of describing sequence: SEQ ID NO: 105:

(2) Information about SEQ ID NO: 106:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 106:

(2) Information about SEQ ID NO: 107:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 107:

(2) Information about SEQ ID NO: 108:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 108:

(2) Information about SEQ ID NO: 109:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 109:

(2) Information about SEQ ID NO: 110:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Method of describing sequence: SEQ ID NO: 110:

(2) Information about SEQ ID NO: 111:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 111:

(2) Information about SEQ ID NO: 112:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Method of describing sequence: SEQ ID NO: 112:

(2) Information about SEQ ID NO: 113:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 113

(2) Information about SEQ ID NO: 114:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 114:

(2) Information about SEQ ID NO: 115:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 115

(2) Information about SEQ ID NO: 116:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 116:

(2) Information about SEQ ID NO: 117:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 117:

(2) Information about SEQ ID NO: 118:

(i) the nature of the sequence:

(A) Length of sequence: 30 amino acids

(B) Type of sequence: amino acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: peptide

(xi) Sequence Description: SEQ ID NO: 118:

Claims (11)

Peptides having SEQ ID NO: 1 or 2 and physiologically acceptable salts and esters thereof: SEQ ID NO: 1 SEQ ID NO: 2 Wherein X represents a proteinaceous or nonproteinaceous amino acid (except glycine), and the amino acids at positions 1, 2, 28, 29 or 30, which are independent of each other, can be part of the sequence individually or together, and the N-terminus Is NR ₁ R ₂ R ₁ represents hydrogen, acetyl, trifluoroacetyl, adamantyl, Fmoc, Z, Boc, Alloc, C ₁ -C ₆ alkyl, C ₂ -C ₈ alkenyl or C ₇ -C ₉ aralkyl, R ₂ represents hydrogen, acetyl, trifluoroacetyl, adamantyl, Fmoc, Z, Boc, Alloc, C ₄ -C ₆ alkyl, C ₂ -C ₈ alkenyl or C ₇ -C ₉ aralkyl) The C-terminus is COR ₃ (where R ₃ is OR ₄ or NR ₄ R ₅ (wherein R ₄ is hydrogen or C ₁ -C ₆ alkyl and R ₅ is hydrogen or C ₁ -C ₆ alkyl) ). The peptide according to claim 1, wherein 1 to 11 of the following modifications are carried out in the amino acid chain: (a) the α-amino acid at position 1 is D-His, Ala, D-Ala, Gly, Lys or D-Lys, of which Ala, Gly or Lys is particularly preferred; or (b) α-amino acid at position 2 is Ser, D-Ser, Thr, D-Thr, Gly, Ala, D-Ala, Ile, D-Ile, Val, D-Val, Leu or D-Leu, preferably Preferably Ser, Thr, Gly, Ala, Ile, Val or Leu; or (c) the α-amanoic acid at position 3 is Glu, D-Glu, Asp, D-Asp, Ala or D-Ala, preferably Glu, Asp or Ala; or (d) the amino acid at position 4 is Ala, D-Ala or β-Ala, preferably Ala; or (e) the α-amino acid at position 5 is Ser, Tyr or Ala; or (f) the α-amino acid at position 6 is Ala, Ile, Val, Leu, Cha or Tyr, preferably Ala, Ile, Val, Leu or Tyr; or (g) the α-amino acid at position 7 is Ala, D-Ala, Tyr, D-Tyr, Ser, D-Ser or D-Thr, preferably Ala, Tyr or Ser; or (h) the α-amino acid at position 8 is Ala, Tyr or Thr; or (i) the α-amino acid at position 9 is Ala, D-Ala, Glu, D-Glu or D-Asp, preferably Ala or Glu; or (j) amino acids at positions 10,11,12,15,16,17,18,19,20,21,24,28,29 are independently mutually proteinaceous or nonproteinaceous D- or L-amino acids, preferably Preferably a proteinaceous L-amino acid; or (k) the α-amino acid at position 13 is a neutral L-amino acid, preferably a neutral proteinaceous L-amino acid; or (l) the α-amino acid at position 14 is a neutral L- or D-amino acid (except L-leucine), preferably Nle, D-Nle, Ala, D-Ala, Ile, D-Ile, Val or D- Substituted by Val, with Ile, double Val or Ala being particularly preferred; or (m) α-amino acid at position 22 is D-Phe, Tyr, D-Tyr, Leu, D-Leu, Val, D-Val, L-Cha, D-Cha, β-1-Nal, β-2 -Nal or β-1-D-Nal, of which Tyr, Leu or Val are preferred; or (n) the α-amino acid at position 23 is Leu, D-Leu, D-Ile, Val, D-Val, L-Cha, D-Cha, Tyr, D-Tyr, Phe or D-Phe, double Leu , Val, Tyr or Phe are preferred; or (o) the α-amino acid at position 25, 26 or 27 is a neutral L- or D-amino acid, preferably a neutral proteinaceous L-amino acid; or (p) The α-amino acid at position 30 is particularly preferably proteinaceous or nonproteinaceous D- or L-amino acid (except glycine), preferably Arg, D-Arg, Tyr or D-Tyr, Arg or Tyr . The peptide according to claim 1 or 2, which contains only proteinaceous amino acids. The peptide of claim 1, wherein the amino acid substitutions occur at position 2 as compared to SEQ ID NO: 1 or 2. 5. 5. The peptide of claim 1, wherein the amino acid substitutions occur at position 14 as compared to SEQ ID NO: 1 or 2. 6. 6. The peptide of claim 1, wherein the amino acid substitutions occur at position 3 as compared to SEQ ID NO: 1 or 2. 7. Having one of SEQ ID NOs: 5, 68, 69, 71, 78-82 or 84-91, wherein the N-terminus is NR ₁ R ₂ (wherein R ₁ is hydrogen, acetyl, trifluoroacetyl, adamantyl, Fmoc, Z, Boc, Alloc, C ₁ -C ₆ alkyl, C ₂ -C ₈ alkenyl or C ₇ -C ₉ aralkyl, R ₂ represents hydrogen, acetyl, trifluoroacetyl, adamantyl, Fmoc, Z, Boc, Alloc, C ₄ -C ₆ alkyl, C ₂ -C ₈ alkenyl or C ₇ -C ₉ aralkyl) Indicate, C-terminus is COR ₃ (wherein R ₃ is OR ₄ or NR ₄ R ₅ (wherein R ₄ is hydrogen or C ₁ -C ₆ alkyl and R ₅ is hydrogen or C ₁ -C ₆ alkyl)) Peptides and physiologically acceptable salts and esters thereof. . The peptide according to any one of claims 1 to 8, which stimulates the release of insulin. A medicament containing a peptide on one species as claimed in any one of claims 1 to 8 in addition to a conventional carrier and auxiliary substance. Use of a peptide as claimed in any one of claims 1 to 8 for the manufacture of a medicament for the treatment of diabetes.